Enzymatic reactions exhibit remarkable selectivity and efficiency, the likes of which are rarely achieved in bench-top chemical reactions. While it is clear that the biochemical prowess of an enzyme arises from the highly ordered structure of its native-folded protein, the detailed mechanism by which it functions has proven elusive. This is because enzymes are not simply static macromolecules that host an active site, as depicted by their crystal structure; rather, they are dynamic molecules whose choreographed motions can gate the transport of substrate to and from the active site and can modulate over time the activity of that site. To develop a mechanistic understanding of how proteins function, it is essential to study this "choreography of life" on the molecular scale. During this reporting period, we have focused on two key areas of technology development: the ability to acquire time-resolved structures of proteins with 150 ps time resolution, and the ability to acquire time-resolved spectra of proteins in crystals with less than 100 fs time resolution. These capabilities allow us to probe protein structural dynamics at an unprecedented level of detail and will help unveil the mechanistic details by which proteins achieve their specific functional goals. Significant milestones have been reached in each of these complementary fronts. For example, in a multinational collaborative effort, we have succeeded in recording a time-resolved structure of carbonmonoxy myoglobin (MbCO) 150 ps after dissociation of a ligand. The model system being studied, MbCO, is a ligand-binding heme protein whose structure and structural evolution effect reversible binding of oxygen as well as discrimination against toxic carbon monoxide. The time-resolved x-ray diffraction pattern reveals the electron density of the protein with atomic (< 2 ?) resolution. The difference electron density map, obtained by subtracting the electron density recorded before and after laser photolysis, reveals correlated positive and negative changes in the vicinity of the active binding site. These changes unveil how the protein responds to ligand dissociation, ligand docking, and ligand expulsion into the surrounding solvent. The data are currently being refined with the assistance of a collaborator, Prof. George Phillips of the University of Wisconsin, in order to model the magnitude and direction of the time-dependent atomic displacements. Our in-house effort to probe protein dynamics spectroscopically has achieved another significant milestone: the ability to record the entire visible-near IR absorbance spectrum of a protein crystal with a single < 100 fs pulse of laser light. This capability, which was first demonstrated in February 20001 and reported at the Biophysical Society Meeting in Boston the same month, paves the way for us to probe protein dynamics in crystals as well as in solution. The unprecedented capabilities of this instrument will allow us to characterize the environment dependence of protein dynamics (e.g., crystal vs. solution) as well as develop photolysis protocols that maximize the yield of photoactivation in protein crystals.